HETEROGENEITY OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED ALKALINE-PHOSPHATASE OF CALF INTESTINE
HETEROGENEITY OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED ALKALINE-PHOSPHATASE OF CALF INTESTINE
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DOI：
10.1111/j.1432-1033.1993.tb18234.x
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发表时间：
1993-10-01
期刊：
影响因子：
--
通讯作者：
FISCHER, W
中科院分区：
文献类型：
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作者：
BUBLITZ, R;ARMESTO, J;FISCHER, W
本发明描述了一种从肠粘膜和食糜中大规模纯化糖基磷脂酰肌醇锚定碱性磷酸酶至均一的方法。这两种酶制剂含有约2 mol脂肪酸/mol亚基，并表现出非常相似的脂肪酸组成与十八烷酸和十六烷酸作为predominant components.No显着差异之间的天然糖基PtdIns锚定和亲水性碱性磷酸酶从两个来源被发现关于K（m），V（最大值），抑制类型和抑制常数的氨基酸L-亮氨酸，L-苯丙氨酸，和L-色氨酸。两种来源的纯化酶分别用磷脂酰肌醇特异性磷脂酶C（PtdIns-PLC）和糖基磷脂酰肌醇磷脂酶D（PLD）处理后产生二酰基甘油和磷脂酸。组分I对应于无锚酶，组分II-V在它们对磷脂酶的敏感性方面不同。馏分II和IV被PtdIns-PLC或PLD作用完全裂解，馏分III几乎50%被PtdIns-PLC裂解，而馏分V是抗性的。这两个馏分对PLD作用的敏感性相当高。脂肪酸分析产生的脂肪酸/碱性磷酸酶亚基的摩尔比为1.78，2.58，2.24，和3.37的馏分II，III，IV，和V，分别。聚集体的糖基PtdIns锚定的碱性磷酸酶的所有馏分在天然PAGE中的Triton X-100的存在下。在Brij 35存在下，通过凝胶色谱法，组分II-V形成稳定的二聚体多重聚集体，并可结合不同量的去污剂。级分II和IV是具有两个分子脂肪酸/亚基的四聚体和八聚体。馏分III是四聚体，带有一个额外的脂肪酸分子，位于二聚体上。级分V是八聚体，含有糖基PtdIns-锚分子，具有三分子脂肪酸/锚分子。额外的脂肪酸残基可能位于肌醇和负责的PtdIns-PLC的敏感性降低。所有测量参数的两种酶的相似性表明，糖基PtdIns-anchored碱性磷酸酶的粘膜被释放到食糜中，而不改变锚分子成分。
A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components.No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regarding K(m), V(max), the type of inhibition and inhibition constants of the amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively.Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatography. Fraction I corresponds to the anchorless enzyme, fractions II-V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns-PLC or PLD action, almost 50% of fraction III is split by PtdIns-PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively.Aggregates of glycosylPtdIns-anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X-100. By gel chromatography in the presence of Brij 35, fractions II-V form stable multiple aggregates of dimers and may bind different amounts of the detergent.These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns-PLC.The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns-anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.